13beta-alkyl-4-gonen-3-ones



are resolved into specific d or I isomers.

United States Patent- This invention relates to the oxidation of steroids and more particularly relates to a microbiological process for the preparation of new hydroxylated steroids which have-valuable therapeutic properties. Specifically, the

new compounds of the presentinvention are useful for their'androgenic activity. Additionally, the inventionencompasses a series of new compounds intermediate to -the newproducts of this invention.

It has now been found possible according to the method of the present inventionto oxidize selected steroids of the gonane series by means of microorganisms and to utilize theproducts thereof as a means of preparing resolved forms of the racemic mixtures of the starting steroid compounds. More specifically, it has been found according to the present invention that if til-steroids of the l3fi-alkyl-4-gonen-3 -one series corresponding to those encompassed within-Formula Itandits mirror image which follows are subjected to microbiological fermentation in a manner hereinafter described, new compounds are obtained which are resolved derivatives of the dl-racemic mixture.

In its broadest aspect, the compounds and the process of the present invention may be illustratively represented by the following sequence of formulae:

wherein'R is 'alkyl; R is selected from the group consisting of hydrogen and acyl; R is selected from-the group consisting of hydrogen, alkyl, alkenyl and alkynyl; and X is selected from the group consisting of H and OH and W, Y, and Z are selected from the group consisting of H, OH and OR wherein R is lower acyl all under the-proviso that when one of W, Y and Z is selected from the group consisting of OH and OR the remainder of this group and X areH; and when X is H, one of W, Y and Z is selected from the group consisting of OH and CR and the remainder of this group are H; and when X is OH, W, Y and Z are H; and when W is OH, the representation of structure of the product is a mirror image of II.

In the foregoing reaction sequence, the starting materials encompassed within Formula I are racemic and In the examples which follow, it will be noted that the products vary iutheir optical activity. Those steroid products substituted in the 1,8-positi-on (i.e., W of Formula II) rotate the plane of polarized light and are characterized as having unnatural configuration. The steroid products substituted at 1013, 6,8 and 110: position, wherein R is other than methyl (i.e., X, Z and Y respectively of Formula II), are d, r dextrorot-atory and are identified as having natural configuration. "Inthe foregoing structures encompassed within II, -,wherein R is methyl and X is OH, the steroid is of unnatural configuration.

The lower alkyl groups/identified by R and R preferably include methyl ethyl, propyl, i-sopropyl, butyl, isobutyl and similar groups. The acyl group identified i: by R is derived from carboxylic. acids preferably having lesslthan: 12 :carbonaatomsfiin saturated oraunsaturated, straight; branched; cyclic or. mixed aliphatic-cyclic :coni figuration. Typical -suitable-esters. include the acetate, propionate,=butyrate, .valerate,lhem-isuccinate, enanthate,

caproate, benzoate and the like. The .alkenyl and-al- .kynyl groups represented by -R :include but are notlimitedto vinyl, ethynyl, propynyland :the like.

3 As istevident," the new compoundsof the'present .in-

vention identified byFormula II are characterized in being hydroxylated according to themethod of the present inventionJat the 15-, l0B-,'6/8-, or Ila-positions, thus ;resulting in d-ihydroxyA-gonen 3eones. It is .to be .understood,-.with respecttothe compounds encompassed withinx-Formula. II, .thationlycnesofw, X, Y or Z-.will-"be hydroxylated or :acylated in a single species, under which circumstance -:-the remaining positions at:.1,:.6,'10 or. 11,

,of .the unique and important features .of the present. in-

vention is thatthe method thereof is'capable .of producing simultaneously, in varying amounts, the various species of hydroxylation, i.-e., the 1,8-, 165-, 10,6- ,and 11ahydroxylated-4-gonen-3-ones encompassed within Formula II.

In the process of the present invention,"hydroxylation of the racemic mixture I is accomplished as has been suggested by the use of a selected microbiological organism. *The'genus Aspergillus has been founduseful for this purpose, with the species-Aspergillus ochraceus being preferred. =More specifically it has been foundthat A. ochraceus NRRL 405 is uniquely-adapted to/efiect .the hydroxylation of the present invention. As has been previously suggested, the introductionof the "hydroxyl group by A. ochraceus occurs predominantly at the 1- position and is 5 in orientation when R is ethyl, propyl or higher alkyl. When R is methyl hydroxylation occurs. predominantly at the 11a position. That is to say,

that inthe fermentation by the fungus the major product willbe of the structure identified .;by compound VII in the reaction sequence which follows. Simultaneously \as is described above and in greater detail hereafter, hydroxyi-ation also occurs at the 65-, and lie-positions. These additional resolved products are graphically set iorth below and identified asvHI, IV and V. The specific compounds are identified merely to suggest typical'steroids-th at are obtainable according to the-method of the present invention.

ethyl-tgonen-EI-one ethyl-4-gonen3-one pounds. I

While the foregoingformulae identify the. l3fl-ethy1- 4-gonen-3-one class for convenience, it must be recognized that the invention encompasses those reactions and reaction products preparableaccording'to the method described falling within the broader class represented by Formula 11 above.

The starting racernirc materials identified by Formula I above, utilized in preparing the new compounds of the present invention, are preferably prepared according to the method described in the copending application Serial Number 228,384, .filedzOc-tober 4, 1962, and Belgian droxy-l3-substituted-4-gonen-3-one (VII). Dehydration with alkali of the product VII results in compound VIII which on treatment with base and dimethyl sulfate results in the 3-methoXy-1,3,5(10)-gonatriene IX. The latter on treatment with lithium and ammonia produces the steroid X which thereafter on treatment with mineral acid according to conventional technique results in the resolved laevo (1) form of compound XI of the starting dl racemic VI.

The novel compounds encompassed within those defined by Formula II are, as has been described, useful for their androgenic properties; and, when it is contemplated that they be used for this purpose, these new compounds are conveniently employed in combination with compatible and therapeutically administrable carriers, diluents, excipients and the like. Suitable liquidcarries include lower alcohols, polyalcohols such as glycol, mineral oil and the like. Additionally, conventional carriers such as methylcellulose, carboxymethylcellulose, starch, sugar and the like can be used. The compositions may, of course, be used in liquid, powder or tablet form as desired. The amount of the new compound administered for the purposes defined may-vary, depending on the requirements,

Patents 623,844, 608,370 and 608,369 and patents cited therein. Therein the synthesis of the compounds falling within the class identified by Formula I are fully described together with the physical characteristics of such com- As has been previously described, treating a selected steroid of the structure offFormulal according to the .method of the present invention results in new steroids which are uniquely resolved. The reaction sequence by which a resolved form of the racemic starting material can be obtained is represented by the following reaction sequence wherein Formulae VII-XI are understood to be mirror images of the structure represented.

(')H OH I 3 HO H H p A. ochraceus A O 0:

(VI) (VII dl-l7B-h droxy-13B- y ethyl-4-gonen-3-one l-138-eth1-3-metho -2 Y xy ethyl-4-gonen-3-one 5(10)-gonadier1-17 3-ol As is evident from the foregoing sequence of reactions, the A. achraceus fermentation process produces the dihybut will normally be administered in an amount of from about 0.5 to about 200 mg./kilo of body weight.

As has been suggested, the fermentative oxidation process of the present invention utilizes a microorganism of the type A. 'ochraceus such as for example NRRL 405 or QM 6731. The foregoing microorganisms are obtainable from known sources such as the Northern Regional Research Laboratories, Peoria, Illinois, or the Quartermaster Culture Collection, Natick, Massachusetts. The microorganisms are, of course, also obtainable by isolating the same from natural sources by standard microbiological techniques.

In carrying out the process of the present invention, aerobic conditions are maintained in the presence of a suitable nutrient medium in temperature in the range of from about 15 C. to about 37 C. in the presence of the fungal species referred to. The reaction to prepare a given quantity of the l-hydroxy steroid is normally complete within a period of from within a few hours to about 80 hours. During the growth of the organism under suitable conditions in the presence of'a steroid unsubstituted at the C-1 position, hydroxylation takes place in that position of steroid ring A.

A. ochmceus NRRL=40S can be grown on a dextrose, peptone, corn steep liquor, salts medium at a temperature of about 28 C. for a period of about 64 hours. Suitable nutrient media which may also be used include a yeast extract-dextrose medium or a malt extract-yeast extract-dextrose medium. Other suitable media which contain the necessary carbon, nitrogen and mineral elements, in the form of carbon include sugars such as glucose, sucrose,

' maltose, Xylose, galactose and so forth; alcohols such as glycerol or mannitol; organic acids such as citric acid, maleic acid, acetic acid and various other natural products containing carbohydrates such as corn starch, corn steep 'liquor, soya bean meal, cotton seed meal and many available materials which have been described heretofore as a source of carbon in steroid fermentation processes. Usually a variety of-carbon sources can be employed in the medium with good results. Suitable sources of nitrogen include a number of the above named materials such as corn steep liquor, soya bean meal, cotton seed meal and various substances such as beef extract, casein, yeast, enzymatically digested proteins, degradation products including peptones, amino acids and many other available proteinaceous materials which have been found to be suitable in supporting the growth of fungi. Inorganic sources of 0 nitrogen include ammonium salts and nitrates which may also be used in the medium as a source of nitrogen to provide a favorable growth medium for the organism.

The mineral requirements of fermentation are usually supplied in the crude materials which are often used as sources of carbon and nitrogen or water that is used in the process. However, it may be advisable to supplement the minerals normally present with added amounts to obtain maximum growth. Cations and anions which may be desirable in added amounts include sodium, potassium, calcium, magnesium, phosphate, sulfate, chloride, cobalt, manganese and various others. The use of elements such as boron, copper, cobalt, molybdenum and chromium is often desirable.

The growth of the organism takes place under aerobic conditions, and aeration in flasks, for example, can be achieved by agitation on a reciprocating or rotary shaker or in bottles or tanks by forcing sterile air through the fermentation mixture. It is desirable that the sterile air be forced through the medium in an amount of from /2 to 2 volumes of air per volume of medium per minute.

Agitation in the bottles or fermenter tanks is provided by mechanical impeller. It is preferable to carry out the process of the present invention at a temperature within the range of about C. to about 37 C.

To obtain large working quantities of the culture A. Ochraceus NRRL 405, the following method may be used. Seven flasks of a corn steep liquor-peptone-dextrose-salts medium, 100 ml. in 500 ml. flasks, are inoculated with a spore suspension prepared from agar slant growth of A. ochracezrs NRRL 405. The flasks are incubated at 28 C. on a rotary shaker, 250 rpm, 2" diameter of rotation, for 64 hours. Ten percent transfers are then made to 12 2-liter flasks, each with 400 ml. of the same medium. After 24 hours of incubation as above, 240 mg. of a selected steroid dissolved in 8 ml. of methanol is added to each flask. Incubation is then continued for 30 hours at which time the flasks are harvested for extraction.

After the growth of the microorganism has taken place to some extent, the steroids are added to the fermentation in solution or finely divided form. One of the preferred methods is to dissolve the steroid in methanol or other water-miscible solvents and add it to the fermentation medium at the desired stage in the process. Although the steroid may precipitate from solution when so added, it is dispersed through the medium as a fine suspension and becomes readily available to the organism for oxidation. The amount of steroid added to the fermentation medium may vary considerably but is generally about to 1 gram per liter of medium.

During the fermentation process, it may be desirable to add anti-foaming agents such as silicones and glyceride oils. These compounds are added from time to time as needed. The fermentation is allowed to proceed for a period of time long enough to achieve maxhnum conversion of the steroid to the l-hydroxy product.

When maximum conversion to the l-hydroxy product is obtained, the desired l-hydroxylated steroid is recovered from the fermentation by extracting with ethyl acetate. The ethyl acetate extracts are concentrated under vacuum to a gum and the latter extracted with boiling hexane. The hexane insoluble solids then contain the desired product which will generally correspond to the structure and configuration of Formula VII. Alternatively, the product obtained by fermentation with A. ochraceus is extracted a number of times such as for example 3 times or more with ethyl acetate and the combined extracts evaporated under vacuum. The residue is then slurried with hot acetone and filtered after a period of from /2 to 2 hours. A portion of the desired product is present as the filtered material and by evaporation of the filtrates additional product is obtained which may be recrystallized from alcohol if desired.

In order to obtain products of the type represented by Fomulae III, IV and V, which are produced simultaneously with the lfi-hydroxy product, but in lesser amount, the recovery is made from the mother liquor remaining after removal of the lli-hydroxy product by crystallization.

Product identification following fermentation is accomplished by chromatography in which a portion of the extract is spotted on filter paper. The paper is subjected to chromatography with a solvent such as for example the bush C. (i.e., Bush, Biochem. J. 50, 370, 1952) solvent system with detection being made by means of ultraviolet light absorption.

The method of the present invention and the products obtainable thereby will be more clearly understood by reference to the specific examples which follow:

Example I A spore suspension of A. ochraceus NRRL 405 grown on an agar slant is utilized to inoculate a 250 ml. flask with 50 ml. of medium of the following composition (German Patent 1,009,627) in which the ingredients are measured in grams, on a gram per liter basis: dextrose 50 g., peptone 20 g., corn steep liquor 5 g., and distilled H O 1,000 ml. To each 50 ml. of the previous mixture, there is added, on a gram per liter basis, 1 ml. of the following composition: Ca(NO -4H O 14.6 g., KH PO 5 g., KCl 10 g., MgSO -7H O 10 g., FeSO.; 1 g., MnSO -H O 3 g. and distilled H 0 1 liter.

The flask is incubated on a rotary shaker, 250 rpm, 28 C. for 64 hours, following which a 10 percent transfer is made to flasks of the same medium. After 24 hours of incubation as above, dl-l7 3-hydroxy-l3/8-ethyl-4-gonen- 3-one, 30 mg. dissolved in 1 ml. of methanol. is added to the flask and the incubation continued for an additional 23 hours. A 5 ml. sample of the whole broth is then equilibrated with 1 ml. of methyl isobutyl ketone and the extract spotted on whatman No. -4 filter paper. The sheet is chromatographed in the Bush C solvent system (toluene, ethyl acetate, methanol, water, '9:1:5:5) and the products formed detected by ultraviolet light absorption. The major product is the l-hydroxy derivative.

Six liters of broth obtained by the fermentation of 3 g. of racemic 17fi-hydroxy-l3B-ethyl-4-gonen-3-one (VI) with A. ochrac'eus is extracted with 1.5 l. of ethyl acetate three times. The extracts are combined and evaporated under vacuum. The residue is slurried with 75 ml. of hot acetone, and the slurry filtered after an hour. The solids thus obtained weigh about 550 mg. By evaporation of the mother liquors, an additional 326 mg of l-1 8,17;9- dihydroxy 13 ,8 ethyl-4-gonen-3-one (VII) is obtained, both fractions being identifiable as homogeneous products by thin-layer chromatography. Further concentration of the mother liquors gives 50 mg. of product.

Recrystallization of the first crop of product from methanol several times gives the analytical sample, MP. 198- 200 C. (Kofler); [oc] +66.9 (1% in ethanol); A 243 my. (6 15.500);

KB: Mm.

2.90 3.03;1., 6.0311, 6.14 6.80 11, 6.94 1, 7.13p., 7.3311, 7.53 7.78% 7.9551,, etc.

Anal.Calc. for C H O C, 74.95; H, 9.27. Found: C, 74.54; H, 8.95.

Exmmple II in... 24.1 m (5 17,000 A553; 5.7%., 5.98 6.15;, etc.

AmrL-Calc. for C H O C, 71.10; H, 8.30. Found: C, 71.18; H, 8.39.

. gonen 3 one,

7 Example III To prepare l-15,l75-dil1ydroxy-17a-ethyl-135-propyl-4 135 propyl 175 hydroxy 17a ethyl- 4-gonen-3-one is fermented with A. ochraceus 'NRRL 405 according to the method previously described in Example I.

1 Example IV To prepare d-65,175-dihydroxy-l7a-propyl-l35-ethyl- 4 gonen 3 one, 135 ethyl 175 hydroxy 17apropyl-4-gonen-3-one is fermented by A. oclzraceus NRRL 405 aspreviously described in Example I.

Example V Mother liquors from which the product of Example I, l-135-ethyl-15,175-dihydroxy-4-gonen-3-one, had been removed by crystallization are combined and dispersed in water. The aqueous dispersion is extracted with petroleurn ether, then with petroleum ether-ether (1 1), to re- 1 move lipids.

The steroidal components are then extracted with ether, chloroform-ether (1:1), and'chloroform. These extracts are combined and evaporated under reduced pressure, yielding 20 g. of mixed crude steroids, which are chromatographed on 800 g. of silica gel, taking 1.5 1. fractions with benzene-ethyl acetate mixtures. The

following fractions are obtained:

Benzene-Ethyl Acetate Ratio Fraction Product Isolated Non-steroidal.

Substrate. 610 mg. III. 3.4 g. mixed III, IV. 810 mg. IV. 240 mg. mixed IV, V.

mg. V.

Elution with pure ethyl acetate affords 900 mg. of an oil from which 70 mg. of VII is recovered.

The material eluated from fractions 66-84 above is obtained after removal of the solvent under vacuum, affording '610 mg. of pure product.

The product, d-135- ethyl-105,175-dihydroxy-4-gonen-3-one (III), is recrystallized from ethyl acetate, giving analytically pure material, M.P.224-227 C., [a] +66.5 (1% chloroform); A 236 ma (6 13,820).

Material eluted from Fractions 1-11-123 above is obtained :by evaporation of the solvent under vacuum. The

crystalline product, d-l35-ethyl-65,175-dihydroxy-4-gonen-3-one (IV), [is further recrystallized from ethyl acetate,

yielding the analytically pure product M.P. 191-195 C., [11] 75.0 (1%, chloroform), k 237.5 ma (e 13,930).

Material obtained from the ethyl acetate washings of the column is recrystallized from ethyl acetate, yielding pure d-135-ethyl 11a,175 dihydroxy-4-gonen-3-one (V), M.P. 215-217 C., A 242 m (a 15,325).

Example VI To prepare 1 15,175 dihydroxy 135 n butyl- 17 a-ethynyl-4-gonen-3-one according to the method of the present invention, 135 n butyl 17a ethynyl 175- hydroxy-4-gonen-3-one is fermented with A. ochraceus.

Example VII According to the method of the previous examples,

I 135 n propyl 17a (1 methallyl) 175 hydroxy- 4-goneu-3-one is fermented with A. ochraceus to produce 1 15,175 dihydroxy 135 n propyl 17a (1methallyl)-4-gonen-3-one.

Example VIII According to the method of the previous examples, 135 n propyl 17a propynyl 175 hydroxy 4- gonen-3-one is fermented with A. ochraceus to produce 81 l 15,175 dihydroxy 135 n propyl 17a propy-nyl- 4-gonen-3-one.

Example IX To prepare l 15,175 dihydroxy 135 n propyl- 17a-vinyl-4-gonen-3-one according to the method of the previous examples, 1 3 5n-propyl-17a-vinyll75-hydroxy- 4-gonen-3-one is fermented with A. ochraceus.

Example X To prepare l-15,'175-dihydroxy-135-pnopy1-4-gonen-3- one, 10 l. of broth obtained by the fermentation with A. ochraceus of 6.5 g. of dl-175-hydroxy-135-propyl-4- gonen-3-one is extracted with 8 l. of ethyl acetate. The mixture is filtered through .Celite filter aid, and the layers separated. The aqueous layer is re-extracted with 8 l. of ethyl acetae. Each ethyl acetate extract is washed with 1 liter of water; the spent mycelium is extracted with 2 l. of ethyl acetate. The three ethyl acetate extracts .are combined and concentrated in vacuum to a dark oil.

The material is dissolved in chloroform-methanol (1:1), filtered, and the solventevaporated. The residue is dissolved in ethyl acetate and ether added. After standing overnight the solids precipitated are filtered, yielding 700 mg. of crystalline product. Recrystallization from methanol several times gives the analytical sample, M.P. 216- 220 C.; [aJ +47.3 (1% in methanol-chloroform, 1:1); x 243.5 ma (a 15,147);

' Anal-Cale. for C H O C, 75.43; H, 9.50. Found: C, 75.16; H, 9.65.

Example XI The method carried out with the starting material and according to the method of Example I in which the medium used consists of yeast-extract 1%, dextrose 1% and distilled water 100 ml.

Example XII 135 ethyl 17a -ethynyl 175 hydroxy 4 gonen- 3-one is fermented with A. ochraceas NRRL 405 to produce l 15,175 dihydroxy 135 ethyl-17a-ethynyl- 4-gonen-3-one.

Example XIII 135,17a diethyl 175 hydroxy 4 gonen 3 one is fermented with A. ochraceus NRRL 405 according to the method of the previous examples to produce d-65,175- dihydroxy-135,17a-diethyl-4-gonen-3 -one.

Example XIV To prepare 1 4 15,175 diacetoxy 1'35 propyl 4- gonen 3 one, 15,175-dihydroxy propyl 4- gonen-S-one is treated with acetic anhydride in the presence of pyridine according to the method of Example VI.

Example XV To prepare d 65,175 diacetoxy 135 ethyl cmethyl 4 gonen 3 one, 65,175 dihydroxy 135- ethyl 17a methyl 4 gonen 3 one is refluxed with two equivalents of acetic anhydride in the presence of ptoluene sul-fonic acid catalyst for about one hour the solution neutralized and the product recovered.

Example XVI 9 Example V11 To prepare d 17 3 acetoxy 13/3 ethyl 10,3 hydroxy-4-gonen-3-one, one hundred milligrams of 11-135- ethyl B17/8 dihydroxy 4 gonen 3 one is dissolved in five milliliters of dry pyridine and one milliliter of acetic anhydride is added. The solution is allowed to stand for fifteen hours, after which time the solvents are removed under vacuum. The residue is repeatedly dissolved in methanol and evaporated under vacuum until the odor of the reagents is removed, and the residue is then recrystallized several times from methanol, yielding the purified d-17B-acetoXy-13fi-ethyl-10flhydroXy-4-gonen-3-one.

Example XVIII To prepare d-l1oz,17/8-diacetoXy-13,3-butyl-4-gonen-3- one, l1oz,17B dihydroxy 13/8 butyl 4 gonen 3 one is treated with acetic anhydride according to the method of Example XV.

While the compounds and method of the present invention have been described with some degree of particularity in the foregoing discussion and in the examples, it is to be understood that nothing contained therein is in anyway to be construed as a limitation on the scope of this invention. The latter is to be limited only by the claims appended hereto.

The invention claimed is:

1. l 1 6,175 di lower acyloxy 13B lower alkyl- 4-gonen-3-one.

2. I- 16175 diacetoxy 13fl ethyl 4 gonen 3- one.

3. l lfi lower acyloxy 135,170 diethyl 17B- hydroxy-4-gonen-3-one.

4. l 1,8,17,6 dihydroxy 13 B lower alkyl 4 gonen- 3-one.

5. l 15,175 dihydroxy 13 3 ethyl 17a ethynyl- 4-gonen-3-one.

6. l- 1,8,1718 dihydroxy 13,8 propyl 4 gonen 3- one.

7. l 1p,17p dihydroxy 13/3 ethyl 4 gonen 3- one.

References Cited by the Examiner UNITED STATES PATENTS 2,602,769 7/ 1952 Murray et al. 51 2,666,016 1/1954 Hechter et a1. 19551 2,708,672 5/1955 Magerlein et a1. 260-39745 2,714,599 8/1955 Konken et a1. 260 397.45

OTHER REFERENCES Pederson et al., J.A.C.S. 78, pp. 1512-13 (1956).

LEWIS GOTTS, Primary Examiner. 

1. L - 1B,17B - DI - LOWER ACYLOXY - 13B - LOWER ALKYL4-GONEN-3-ONE. 